Junyang Xian
PhD student in Department of Chemistry
Research field: MALD/I technique
Attentative adivsor: Dr. Owens
1. Introduction of MALD/I
Scientists were finding out effective techniques for determining the molecular masses of biopolymers such as carbohydrates and proteins. Traditional mass spectrometric methods were exhibited very suitable for testing compounds with low molecular masses, however, when it goes to large compounds, it becomes not so useful as we expect.[1] Thanks to the development of Matrix Associated Laser Desorption Ionization (MALD/I) technique, the structure of large molecules such as polymers, biomacromolecules, can be more characterized. MALD/I can supply the information of repeat units of polymers, the molecule mass distribution and the end functional groups. But due to complexity of both physical and chemical in these systems, even MALD/I has been widely utilized in application], fundamental understanding of this experiment is developing slowly, many work based on empirical approach. As we known, relying on empirical work will be less and less profitable in the future. Accordingly, in order to purposefully design an efficient and profitable MALDI experiment, more and more efforts should be taken in developing understanding of the mechanism of this technique.
With the objection of achieve a successful MALD/I experiment, a serious of variable conditions should be considered, which include matrix choice, analyte physical and chemical properties, concentrations, preparation method, and laser characteristics (wavelength, spatial, mode and temporal pulse shape, local environment (ambient pressure or substrate temperature), and ion extraction method.[2] The most important concept includes the ionization of matrix molecules, together with charge transfer between matrix ions and analytes. This essay will introduce the research of the last part, ionization mechanisms, which play an critical role in MALD/I technique.
2. The history of study in MALD/I ionization
MALD/I ionization has been studied for a long time, since 1983, Hillenkamp’s group first reported the characterics of “true” laser desorption. After that, there were an large amount of research had been focused on the development of this field, that set a foundation for the coming out of MALD/I in the mid 1980s.[3-5]It took not a period of long time since several significant reviews had been published, which made remarkable contribution to the fundamental understanding of the ionization mechanism of MALD/I. Ehring, Karas, and Hillenkamp groups regarded a highly excited matrix was a key intermediate step for all MALD/I process, which induced the outcome of modern MALD/I.[6] Liao and Allison and their co-workers followed this opinion, and found that MALD/I process probably cause protonated and sodiated adduct.[7] That work made a specific contribution in reorganizations of the central role of ion-molecule reaction in MALD/I desorption plume, and the relevance of the gas phase thermodynamics of these reactions to the observed mass spectra. That was the primitive recoganization of MALD/I.
During 1990s, research was continuously improved in this area. Karas, Bahr and Stahi Zeng and their co-workers enhanced proton transfer reactions of the matrix with analyte. figured out desorption, ionization and analyte fragmentation were aspects of matrix function in 1996. The first review focused on MALD/I ionization mechanisms published in 1998.[9] In 2003 an review had summary laser ablation of molecular substrates. Zhigilei and Garrison groups provided an important microscopic view of the desorption/ablation event, induced by molecular dynamics work of MALD/I.[9]
It is under continuously debate that whether the matrix ions are formed through multiphoton excitation, annihilation/energy pooling processes, excited-state thermoionic emission process, or whether they are preformed in crystal.[1--18]
For study of matrix ionization, analysis of photoelectrons and ion pairs plays an important role. Frankevich and Knochenmuss’s group found that the substrate is closely associated with the source of photoelectrons.[19,20] Yi-Sheng Wang and his co-workers found that the threshold laser fluence (Fth) of forming photoelectrons was under that of matrix ions, in their studies, the difference in Fth between photoelectron and ino formation grows up in a large extend in the sequence THAP < SA < DHB. It is logical that different ionization pathways of every matrix cause this difference.[21-23]
The [[#|energy cost]] by chemical reaction should also be considered in MALD/I. process. Take for instance the increase of system temperance for instance, which is an essential factor in in MALD/I process. There are different results obtained from infared emission, thermometer molecules survival yield, thermometer molecules, molecules internal energy as well as theoretical simulations. [9,24-30] These chemical reactions can absorb energy in MALD/I process and compete with other reactions, then finally influence ion productivity. The percentages of these chemical reactions take place when come to dynamic balances depend on the properties the matrix molecule as well as excitation method. Yi-Sheng Wang and co-workers reported that Photoionizaion is important if matrix molecule highly absorb cross section or lifetime of an excited state which is at the nanosecond range, take the frequently used matrix, DHB molecule, fro example. If significant energy can be converted to thermal energy, it will allow thermal ionization be induced in ground state by using near UV laser.- They have put variety of matrix molecules in their experiment, and found THAP molecule can be an ideal example, whose excited state lifetime or significant decomposition does not take long when excited by a near UV laser. SA molecule should be considered as another category, even it does not take an excited-state lifetime at the nanosecond rand, photonization is still play an important role owing to the highly absorb cross section. They also supported that the exact ionization mechanism relies on the chemical properties of matrix molecules and the condition of excitation.[31]
3. Cluster model
The cluster model was brought up and largely developed by Karas and his co-workers The cluster model can also see as the “lucky survivors” model.[17] The positive charged ions in the solid matrix will encounter largely neutralization by electron in the plume.[2] , the left ions in this process are important for us to study, which called luck survivors, the sketch of this process is shown in Figure 1.
Figure 1 Process of cluster models in MALD/I ionization.[2]
At first, the ions are preformed in solutions, after ablation expansion, they will contained in clusters which ablated from the original solid material. In some of the clusters, charge could be transfer from matrix to analyte. For the clusters which are charged positively, after they encounter protonation, the cluster may release the ion. The multiply charged analytes are divide into “hard”(only low anlyte charge states perfom) and “soft”(loss of neutral matrix), and each of them may release different ions.
4. Two-step framework and primary and secondary mechanism
It is commonly agreed that MALD/I ionization process will encounter two=steps, that is called two-step framework of MALD/I ionization. The first step is considered as primary ion formation or separation, and then the second step is regarded as ion molecule reactions in the desorption/ablation plume, in this step, secondary ions are formed and then will attach to the detector finally. The authenticity of the first step mechanism is still under controversial, but the second step mechanism is generally agreed by scientists. It becomes more and more accepted that the plume in MALD/I process is always has sufficient density and life span for the matrix and analyte species to apply conventional kinetics and thermodynamics reactions. Both of these two step are a result of physical characteristics of the MALD/I process, which is very important for the fundamentally understanding of the mechanism MALD/I ionization.[2]
5. Multiphoton Ionization
Molecule multiphoton ionization (MPI) is most accepted mechanism for MALD/I ionization under laser excitation of an absorbing organic material. This process has been supported by many authors which they considered highly probably happen in MALD/I ionization process.[8] Ehring, Karas, and Hillenkamp proposed that photonized matrix radicals are important providers of other MALD/I ions,[6] as shown in Figure 2.
Figure 2 [[#|Schematic diagram]] of photoionization and subsequent reaction pathways in MALD/I, energy pooling (A), sequential two-photon excitation (B), simultaneous two-photon excitation (C), all result in a highly excited intermediate, which was considered as emitting one electron in the basic ionization step.[8]
As mentioned above, the whole process divides into two parts. The first part is primary ion formation, which includes excitation and photoionization. The second part is secondary reactions, which is photochemical reactions. We will talk about these two steps more specifically as follows.
As shown in Figure 1, the first step of MALD/I ionization is excitation. There are three pathways of excitation. The first pathway is energy pooling, Photoexcation in matrix molecules is a widely accepted concept which has been extensively described in the literature, especially for one of the most common-used matrix, 2,5-dihydroxybenzoic acid (DHB). Yi-Sheng Wang and co-workers reported that for DHB, sinapinic acid (SA), and trihydroxyacetopheone (THAP), the primary ionization reactions are proceed by the photoelectrons that ejected by photoionization. Equations 1-3 describe the three fundamental pathways of initial ionization induced by laser photos in Figure 1.[23]
The first equation is photoionization, a sequential multiphoton absorption or annihilation mechanism may involve in this step.[7,8,14,-16] The existence of radical ions in many matrixes with low laser fluence shows the evidence for this equation.[6,13,33] Yi-Sheng Wang’s group proved that there is a powerful evidence of DHB molecule was received for equation 1.[20-22].The second and third equation are proved by protonated and deprotonated molecules as major products in MALD/I conditions. But proton disproportionation in the excited state has been regarded as unfavorable for frequently used matrixes.[9] Further study is necessary to figure out whether these equations could happen.
Pooling was one of sorts of energy concentration possibility proposed in early study. It is a process that the electronic excitation energy of two adjacentmolecules could be redistributed. However, two photons from N2 or tripled Nd:YAG lasers are not enough to ionize free many matrix molecules such as DHB.[34] If the two nearby molecules are both individually excited to the first excited singlet state (S1) through photoexcitation, and their excited states have strong interaction owing to wavefunction overlap, the couple system could redistribute the energy in different ways, one molecule becomes S0 state, and the other goes to Sn state, but at this step, the energy is not enough for ionization, as shown in Figure 3. At the second step, if the excited Sn state molecule meets a molecule which in S1 state, the energy of both of the could be redistribute again, the former molecule raises up to ionization state, the latter goes down to ground state S0, thus, this process can provide sufficient energy for formation of ions from matrix molecules.[2] Figure 3 Pooling processes of matrix molecule excited states.[2]
The interaction between different molecule which stay at various excited state is the key aspect of this model. The process of this model includes, which includes hopping, pooling, quenching as well as recombination are all second order, their rates are strongly depends on the intermolecular collision rates.[2]
The ionization potential (IP) of most matrix molecules stay in 3-photon region of typical MALD/I lasers, correspond to that of DHB molecule, stays at 8.054eV.[34] Because most of matrices are aromatic compounds, which have conjugated π-systems with similar size, accordingly, their IP are also not quite different.[2] IPs can even be calculated with errors lower than 0.2eV.[33,34,35] As we know, the IP of free matrix is high, but when IPs of clusters of matrix and analyte can be largely deduced compared with that of free matrix.[38] This reduction results from matrix-analyte interaction. Particularly, when there are proton or electron groups, charge transfer between nearby molecules can facilitate phtoionization of the matrix-analyte system
There are several authors proposed this explanation. Kinsel and his co-workers reported this effect in clusters.[39-42] They also found that for DHB-proline molecules, the post ionization fragmentation can protonated analytes, which provides another pathway in MALD/I ionization process. [39,41,42]Correspondingly, the MALD/I process in practical was considered as the same as three multiple pathways above. A relationship between the size of matrix molecules and its ionization potential (IP) has been found. It has been reported that as increasing the number of DHB-(proline)n clusters, the levels of IP of them are continuously decreasing, both theoretically and experimentally.[43] But due to lackinreliable simulation of large DHB clusters, this conclusion has not been confirmed in practice. It has already been determined by Knochenmuss’s group that ionization (IP) of gaseous (DHB)1-10 clusters through the first equation was roughly balances at 7.85eV, as shown in figure 4,[33,43] the photonization threshold decreases as increasing of cluster size of DHB molecule, higher than the two frequently used later wavelengths (337nm=3.68eV, 335nm=3.48eV). This trend as the increasing of cluster size will reach a plateau, accordingly, as the size of cluster increasing to higher sizes, its influence on the IP of molecules should become lower.[43] Figure 4 The relationship between cluster size of DHB molecule and its photoionzaion threshold.[43]
IPs are regarded as relevant to the ability of electron transfer. Reduction in IP of matrix ions will lead them to present lowering in ability to ionize analyte. Accordingly, free matrix ions are more probably to play a part in secondary electron transfer reactions than cluster. Interestingly, the IPs of matrix/analyte cluster can also be lowering compared to the free matrix ions. This effect is more dramatic and important than the former one. Accordingly, the decreasing of IP in clusters could be very significant in primary mechanism.
Yi-Sheng Wang’s group supported that solid matrix molecules with large cluster sizes were probably the main origin of photoelectrons. Accordingly, solid matrix molecules with larger sizes contribute more in primary ionization process than the smaller one, no matter considering from the aspect of IP or the amount of protonelectrons formed by photoionizaion.[33]
6. Excited-State Proton Transfer
Since the ortigins of MALD/I, excited-state proton transfer (ESPT) has been considered as the most supported MALD/I ionization model after the process of photoionization, This model is attractive due to it can occur in the first electronic excited state, thus, it is one-photon event with excitation.. When a singly matrix molecules is excited, it will become more acidic than its ground state. Accordingly, the adjacent analyte or other matrix molecules in ground state probably accept a labile proton. The process is shown as follows:[8]
M + hv → M*
M* + A → (M–H)- + AH+
M* + M →(M–H)- + MH+
A large amoun of excellent UV matrices are structurally similar to ESPT species like salicylic acid (SA). The hydroxyl group of SA is ortho to the carbonyl group (CO, COOH, CONH2, etc.). As shown in Figure 5, in excited SA molecule, the hydroxyl proton is transferred to the carbonyl oxygen.
Figure 5 Intramolecular ESPT of SA[45]
However, ESPT is highly dependent on whether its environment can stabilize charge separation. As we known, ESPT processes could only take place in water or amine environment, where matrix-analyte complexes are predisposed to proton transfer through highly asymmetric hydrogen bonds which could stabilize neighbor substituents. Accordingly, ESPT species have not been widely used in . MALD/I and finding direct indicators of ESPT like DHB molecule has not been successful both in solution and clusters. ESPT is too costly for a single near-UV proton without assistance of intra- or intermolecular.[43]
7. Secondary processes
After the primary ionization, the MALD/I events will encounter the second step. All the matrix and analyte species, which include clusters or particles, will become free to be analyzed. Even some of the matrix or analyte molecules could be ionized in the first step, but most of the matrix molecules, which should be largely excess, are neutral, accordingly, the ions will encounter the collisions with neutral matrix. It might involve electron transfer in the process as below:[2]
m*+ + A → m + A*+
It is also be considered that proton transfer or cation transfer could be include in this event. The consequence of the thermodynamic model of this process has been successfully examined. Kinsel et al found that ln (Analyte/Matrix) of CHCA to series of amino has linear relationship with the basicity of amino acid, suggesting that the proton transfer reaction in this system reached equilibrium in the plume,[2] as shown in Figrue 6.
Figure 6 Equilibrim polt of the MALD/I ion signals of the amino acids G. A. V. L. and F in the matrix CHCA.[2]
In addition, there are other less direct results of the thermodynamic model of the plume which have been successfully examined, but before testing of these it is necessary to consider the categories of ions and reactions which should be expected in the plume. We will not talk about these aspect in this essay.
8. Summary
MALD/I is service as an advanced technique of testing the masses and structures of biopolymers compared to the traditional ones. Ionization of MALD/I plays an important role in this process. Cluster model is a famous model in MALD/I ionization. Both of the matrix and analyte will go through an expansion process in the plume and become cluster with ions, and then some of them react and perform different ions. MALD/I ionization is considered as two-step framework, with increasingly agreement by scientists. The first step is primary ion formation, It becomes more and more accepted that the plume in MALD/I process is always has sufficient density and life span for the matrix and analyte species to apply conventional kinetics and thermodynamics reactions, which is the second step in MALD/I ionization process . Both of these two step are a result of physical characteristics of the MALD/I process, which is very important for the fundamentally understanding of the mechanism MALD/I ionization. Molecule multiphoton ionization (MPI) is the most accepted mechanism for MALD/I ionization. The whole process divides into two parts. The first part is primary ion formation, which includes excitation and photoionization. The second part is secondary reactions, which is photochemical reactions. Pooling was one of sorts of energy concentration possibility proposed in early study. It is a process that the electronic excitation energy of two adjacent molecules could be redistributed. Ionization potential is another factor should be consider in MALD/I ionization ,as increasing the number of DHB-(proline)n clusters, the levels of IP of them are continuously decreasing, both theoretically and experimentally. Excited-state proton transfer has been considered as the most supported MALD/I ionization model after the process of photoionization, This model is attractive due to it can occur in the first electronic excited state, thus, it is one-photon event with excitation.. When a singly matrix molecules is excited, it will become more acidic than its ground state.
To sum up, fundamental understanding of MALD/I ionization has developed this process from a totally empirical one to a more predictive one. Those understandings could be used in practical application, which is benefit for design more effective MALD/I experiment. However, we should admit that even though the understanding of MALD/I ionization has been developed, from totally empirical to practical application, but there is still a lot leave us to learn about this complex technique.
Reference
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The Process of MALD/I Ionization
Junyang Xian
PhD student in Department of Chemistry
Research field: MALD/I technique
Attentative adivsor: Dr. Owens
1. Introduction of MALD/I
Scientists were finding out effective techniques for determining the molecular masses of biopolymers such as carbohydrates and proteins. Traditional mass spectrometric methods were exhibited very suitable for testing compounds with low molecular masses, however, when it goes to large compounds, it becomes not so useful as we expect.[1] Thanks to the development of Matrix Associated Laser Desorption Ionization (MALD/I) technique, the structure of large molecules such as polymers, biomacromolecules, can be more characterized. MALD/I can supply the information of repeat units of polymers, the molecule mass distribution and the end functional groups. But due to complexity of both physical and chemical in these systems, even MALD/I has been widely utilized in application], fundamental understanding of this experiment is developing slowly, many work based on empirical approach. As we known, relying on empirical work will be less and less profitable in the future. Accordingly, in order to purposefully design an efficient and profitable MALDI experiment, more and more efforts should be taken in developing understanding of the mechanism of this technique.
With the objection of achieve a successful MALD/I experiment, a serious of variable conditions should be considered, which include matrix choice, analyte physical and chemical properties, concentrations, preparation method, and laser characteristics (wavelength, spatial, mode and temporal pulse shape, local environment (ambient pressure or substrate temperature), and ion extraction method.[2] The most important concept includes the ionization of matrix molecules, together with charge transfer between matrix ions and analytes. This essay will introduce the research of the last part, ionization mechanisms, which play an critical role in MALD/I technique.
2. The history of study in MALD/I ionization
MALD/I ionization has been studied for a long time, since 1983, Hillenkamp’s group first reported the characterics of “true” laser desorption. After that, there were an large amount of research had been focused on the development of this field, that set a foundation for the coming out of MALD/I in the mid 1980s.[3-5]It took not a period of long time since several significant reviews had been published, which made remarkable contribution to the fundamental understanding of the ionization mechanism of MALD/I. Ehring, Karas, and Hillenkamp groups regarded a highly excited matrix was a key intermediate step for all MALD/I process, which induced the outcome of modern MALD/I.[6] Liao and Allison and their co-workers followed this opinion, and found that MALD/I process probably cause protonated and sodiated adduct.[7] That work made a specific contribution in reorganizations of the central role of ion-molecule reaction in MALD/I desorption plume, and the relevance of the gas phase thermodynamics of these reactions to the observed mass spectra. That was the primitive recoganization of MALD/I.
During 1990s, research was continuously improved in this area. Karas, Bahr and Stahi Zeng and their co-workers enhanced proton transfer reactions of the matrix with analyte. figured out desorption, ionization and analyte fragmentation were aspects of matrix function in 1996. The first review focused on MALD/I ionization mechanisms published in 1998.[9] In 2003 an review had summary laser ablation of molecular substrates. Zhigilei and Garrison groups provided an important microscopic view of the desorption/ablation event, induced by molecular dynamics work of MALD/I.[9]
It is under continuously debate that whether the matrix ions are formed through multiphoton excitation, annihilation/energy pooling processes, excited-state thermoionic emission process, or whether they are preformed in crystal.[1--18]
For study of matrix ionization, analysis of photoelectrons and ion pairs plays an important role. Frankevich and Knochenmuss’s group found that the substrate is closely associated with the source of photoelectrons.[19,20] Yi-Sheng Wang and his co-workers found that the threshold laser fluence (Fth) of forming photoelectrons was under that of matrix ions, in their studies, the difference in Fth between photoelectron and ino formation grows up in a large extend in the sequence THAP < SA < DHB. It is logical that different ionization pathways of every matrix cause this difference.[21-23]
The [[#|energy cost]] by chemical reaction should also be considered in MALD/I. process. Take for instance the increase of system temperance for instance, which is an essential factor in in MALD/I process. There are different results obtained from infared emission, thermometer molecules survival yield, thermometer molecules, molecules internal energy as well as theoretical simulations. [9,24-30] These chemical reactions can absorb energy in MALD/I process and compete with other reactions, then finally influence ion productivity. The percentages of these chemical reactions take place when come to dynamic balances depend on the properties the matrix molecule as well as excitation method. Yi-Sheng Wang and co-workers reported that Photoionizaion is important if matrix molecule highly absorb cross section or lifetime of an excited state which is at the nanosecond range, take the frequently used matrix, DHB molecule, fro example. If significant energy can be converted to thermal energy, it will allow thermal ionization be induced in ground state by using near UV laser.- They have put variety of matrix molecules in their experiment, and found THAP molecule can be an ideal example, whose excited state lifetime or significant decomposition does not take long when excited by a near UV laser. SA molecule should be considered as another category, even it does not take an excited-state lifetime at the nanosecond rand, photonization is still play an important role owing to the highly absorb cross section. They also supported that the exact ionization mechanism relies on the chemical properties of matrix molecules and the condition of excitation.[31]
3. Cluster model
The cluster model was brought up and largely developed by Karas and his co-workers The cluster model can also see as the “lucky survivors” model.[17] The positive charged ions in the solid matrix will encounter largely neutralization by electron in the plume.[2] , the left ions in this process are important for us to study, which called luck survivors, the sketch of this process is shown in Figure 1.
Figure 1 Process of cluster models in MALD/I ionization.[2]
At first, the ions are preformed in solutions, after ablation expansion, they will contained in clusters which ablated from the original solid material. In some of the clusters, charge could be transfer from matrix to analyte. For the clusters which are charged positively, after they encounter protonation, the cluster may release the ion. The multiply charged analytes are divide into “hard”(only low anlyte charge states perfom) and “soft”(loss of neutral matrix), and each of them may release different ions.
4. Two-step framework and primary and secondary mechanism
It is commonly agreed that MALD/I ionization process will encounter two=steps, that is called two-step framework of MALD/I ionization. The first step is considered as primary ion formation or separation, and then the second step is regarded as ion molecule reactions in the desorption/ablation plume, in this step, secondary ions are formed and then will attach to the detector finally. The authenticity of the first step mechanism is still under controversial, but the second step mechanism is generally agreed by scientists. It becomes more and more accepted that the plume in MALD/I process is always has sufficient density and life span for the matrix and analyte species to apply conventional kinetics and thermodynamics reactions. Both of these two step are a result of physical characteristics of the MALD/I process, which is very important for the fundamentally understanding of the mechanism MALD/I ionization.[2]
5. Multiphoton Ionization
Molecule multiphoton ionization (MPI) is most accepted mechanism for MALD/I ionization under laser excitation of an absorbing organic material. This process has been supported by many authors which they considered highly probably happen in MALD/I ionization process.[8] Ehring, Karas, and Hillenkamp proposed that photonized matrix radicals are important providers of other MALD/I ions,[6] as shown in Figure 2.
Figure 2 [[#|Schematic diagram]] of photoionization and subsequent reaction pathways in MALD/I, energy pooling (A), sequential two-photon excitation (B), simultaneous two-photon excitation (C), all result in a highly excited intermediate, which was considered as emitting one electron in the basic ionization step.[8]
As mentioned above, the whole process divides into two parts. The first part is primary ion formation, which includes excitation and photoionization. The second part is secondary reactions, which is photochemical reactions. We will talk about these two steps more specifically as follows.
As shown in Figure 1, the first step of MALD/I ionization is excitation. There are three pathways of excitation. The first pathway is energy pooling, Photoexcation in matrix molecules is a widely accepted concept which has been extensively described in the literature, especially for one of the most common-used matrix, 2,5-dihydroxybenzoic acid (DHB). Yi-Sheng Wang and co-workers reported that for DHB, sinapinic acid (SA), and trihydroxyacetopheone (THAP), the primary ionization reactions are proceed by the photoelectrons that ejected by photoionization. Equations 1-3 describe the three fundamental pathways of initial ionization induced by laser photos in Figure 1.[23]
Photoionization M+nhv → Mn++e- (1)
Electron disproportionation 2M+nhv → M●++M●- (2)
Proton disproportionation 2M+nhv → [M + H]- + [M – H]+ (3)
M stands for the matrix molecules.
The first equation is photoionization, a sequential multiphoton absorption or annihilation mechanism may involve in this step.[7,8,14,-16] The existence of radical ions in many matrixes with low laser fluence shows the evidence for this equation.[6,13,33] Yi-Sheng Wang’s group proved that there is a powerful evidence of DHB molecule was received for equation 1.[20-22].The second and third equation are proved by protonated and deprotonated molecules as major products in MALD/I conditions. But proton disproportionation in the excited state has been regarded as unfavorable for frequently used matrixes.[9] Further study is necessary to figure out whether these equations could happen.
Pooling was one of sorts of energy concentration possibility proposed in early study. It is a process that the electronic excitation energy of two adjacentmolecules could be redistributed. However, two photons from N2 or tripled Nd:YAG lasers are not enough to ionize free many matrix molecules such as DHB.[34] If the two nearby molecules are both individually excited to the first excited singlet state (S1) through photoexcitation, and their excited states have strong interaction owing to wavefunction overlap, the couple system could redistribute the energy in different ways, one molecule becomes S0 state, and the other goes to Sn state, but at this step, the energy is not enough for ionization, as shown in Figure 3. At the second step, if the excited Sn state molecule meets a molecule which in S1 state, the energy of both of the could be redistribute again, the former molecule raises up to ionization state, the latter goes down to ground state S0, thus, this process can provide sufficient energy for formation of ions from matrix molecules.[2]
Figure 3 Pooling processes of matrix molecule excited states.[2]
The interaction between different molecule which stay at various excited state is the key aspect of this model. The process of this model includes, which includes hopping, pooling, quenching as well as recombination are all second order, their rates are strongly depends on the intermolecular collision rates.[2]
The ionization potential (IP) of most matrix molecules stay in 3-photon region of typical MALD/I lasers, correspond to that of DHB molecule, stays at 8.054eV.[34] Because most of matrices are aromatic compounds, which have conjugated π-systems with similar size, accordingly, their IP are also not quite different.[2] IPs can even be calculated with errors lower than 0.2eV.[33,34,35] As we know, the IP of free matrix is high, but when IPs of clusters of matrix and analyte can be largely deduced compared with that of free matrix.[38] This reduction results from matrix-analyte interaction. Particularly, when there are proton or electron groups, charge transfer between nearby molecules can facilitate phtoionization of the matrix-analyte system
There are several authors proposed this explanation. Kinsel and his co-workers reported this effect in clusters.[39-42] They also found that for DHB-proline molecules, the post ionization fragmentation can protonated analytes, which provides another pathway in MALD/I ionization process. [39,41,42]Correspondingly, the MALD/I process in practical was considered as the same as three multiple pathways above. A relationship between the size of matrix molecules and its ionization potential (IP) has been found. It has been reported that as increasing the number of DHB-(proline)n clusters, the levels of IP of them are continuously decreasing, both theoretically and experimentally.[43] But due to lackinreliable simulation of large DHB clusters, this conclusion has not been confirmed in practice. It has already been determined by Knochenmuss’s group that ionization (IP) of gaseous (DHB)1-10 clusters through the first equation was roughly balances at 7.85eV, as shown in figure 4,[33,43] the photonization threshold decreases as increasing of cluster size of DHB molecule, higher than the two frequently used later wavelengths (337nm=3.68eV, 335nm=3.48eV). This trend as the increasing of cluster size will reach a plateau, accordingly, as the size of cluster increasing to higher sizes, its influence on the IP of molecules should become lower.[43]
Figure 4 The relationship between cluster size of DHB molecule and its photoionzaion threshold.[43]
IPs are regarded as relevant to the ability of electron transfer. Reduction in IP of matrix ions will lead them to present lowering in ability to ionize analyte. Accordingly, free matrix ions are more probably to play a part in secondary electron transfer reactions than cluster. Interestingly, the IPs of matrix/analyte cluster can also be lowering compared to the free matrix ions. This effect is more dramatic and important than the former one. Accordingly, the decreasing of IP in clusters could be very significant in primary mechanism.
Yi-Sheng Wang’s group supported that solid matrix molecules with large cluster sizes were probably the main origin of photoelectrons. Accordingly, solid matrix molecules with larger sizes contribute more in primary ionization process than the smaller one, no matter considering from the aspect of IP or the amount of protonelectrons formed by photoionizaion.[33]
6. Excited-State Proton Transfer
Since the ortigins of MALD/I, excited-state proton transfer (ESPT) has been considered as the most supported MALD/I ionization model after the process of photoionization, This model is attractive due to it can occur in the first electronic excited state, thus, it is one-photon event with excitation.. When a singly matrix molecules is excited, it will become more acidic than its ground state. Accordingly, the adjacent analyte or other matrix molecules in ground state probably accept a labile proton. The process is shown as follows:[8]
M + hv → M*
M* + A → (M–H)- + AH+
M* + M →(M–H)- + MH+
A large amoun of excellent UV matrices are structurally similar to ESPT species like salicylic acid (SA). The hydroxyl group of SA is ortho to the carbonyl group (CO, COOH, CONH2, etc.). As shown in Figure 5, in excited SA molecule, the hydroxyl proton is transferred to the carbonyl oxygen.
Figure 5 Intramolecular ESPT of SA[45]
However, ESPT is highly dependent on whether its environment can stabilize charge separation. As we known, ESPT processes could only take place in water or amine environment, where matrix-analyte complexes are predisposed to proton transfer through highly asymmetric hydrogen bonds which could stabilize neighbor substituents. Accordingly, ESPT species have not been widely used in . MALD/I and finding direct indicators of ESPT like DHB molecule has not been successful both in solution and clusters. ESPT is too costly for a single near-UV proton without assistance of intra- or intermolecular.[43]
7. Secondary processes
After the primary ionization, the MALD/I events will encounter the second step. All the matrix and analyte species, which include clusters or particles, will become free to be analyzed. Even some of the matrix or analyte molecules could be ionized in the first step, but most of the matrix molecules, which should be largely excess, are neutral, accordingly, the ions will encounter the collisions with neutral matrix. It might involve electron transfer in the process as below:[2]
m*+ + A → m + A*+
It is also be considered that proton transfer or cation transfer could be include in this event. The consequence of the thermodynamic model of this process has been successfully examined. Kinsel et al found that ln (Analyte/Matrix) of CHCA to series of amino has linear relationship with the basicity of amino acid, suggesting that the proton transfer reaction in this system reached equilibrium in the plume,[2] as shown in Figrue 6.
Figure 6 Equilibrim polt of the MALD/I ion signals of the amino acids G. A. V. L. and F in the matrix CHCA.[2]
In addition, there are other less direct results of the thermodynamic model of the plume which have been successfully examined, but before testing of these it is necessary to consider the categories of ions and reactions which should be expected in the plume. We will not talk about these aspect in this essay.
8. Summary
MALD/I is service as an advanced technique of testing the masses and structures of biopolymers compared to the traditional ones. Ionization of MALD/I plays an important role in this process. Cluster model is a famous model in MALD/I ionization. Both of the matrix and analyte will go through an expansion process in the plume and become cluster with ions, and then some of them react and perform different ions. MALD/I ionization is considered as two-step framework, with increasingly agreement by scientists. The first step is primary ion formation, It becomes more and more accepted that the plume in MALD/I process is always has sufficient density and life span for the matrix and analyte species to apply conventional kinetics and thermodynamics reactions, which is the second step in MALD/I ionization process . Both of these two step are a result of physical characteristics of the MALD/I process, which is very important for the fundamentally understanding of the mechanism MALD/I ionization. Molecule multiphoton ionization (MPI) is the most accepted mechanism for MALD/I ionization. The whole process divides into two parts. The first part is primary ion formation, which includes excitation and photoionization. The second part is secondary reactions, which is photochemical reactions. Pooling was one of sorts of energy concentration possibility proposed in early study. It is a process that the electronic excitation energy of two adjacent molecules could be redistributed. Ionization potential is another factor should be consider in MALD/I ionization ,as increasing the number of DHB-(proline)n clusters, the levels of IP of them are continuously decreasing, both theoretically and experimentally. Excited-state proton transfer has been considered as the most supported MALD/I ionization model after the process of photoionization, This model is attractive due to it can occur in the first electronic excited state, thus, it is one-photon event with excitation.. When a singly matrix molecules is excited, it will become more acidic than its ground state.
To sum up, fundamental understanding of MALD/I ionization has developed this process from a totally empirical one to a more predictive one. Those understandings could be used in practical application, which is benefit for design more effective MALD/I experiment. However, we should admit that even though the understanding of MALD/I ionization has been developed, from totally empirical to practical application, but there is still a lot leave us to learn about this complex technique.
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